Prosthetic Grafts

ABSTRACT

An improved prosthetic graft for the bypass, replacement or repair of vessels and organs that are in contact with blood flow is disclosed. The prosthetic graft includes a porous prosthetic implant and adherent cells adhered to the outer surface of the implant. The adherent cells are transfected with at least one recombinant nucleic acid molecule encoding at least one protein that enhances patency of the graft. The prosthetic graft has a long-term patency and success rate that is superior to other previously described prosthetic grafts designed for such use. Also disclosed are methods of making and using such a graft.

FIELD OF THE INVENTION

The present invention relates to prosthetic grafts which are used tocontain blood flow in vivo.

BACKGROUND OF THE INVENTION

Diseases of the major circulatory and renal organs and vessels havecreated a need for prosthetic grafts to bypass, repair and/or replacethe function of the diseased organs and vessels. Such grafts shouldideally be non-immunogenic, non-calcific, and readily capable ofrecreating or reestablishing the natural blood contact interface of theorgan or vessel to be replaced or repaired. Complications that haveinhibited the widespread use of prosthetic grafts in organs and vesselsin contact with blood include: (1) intimal hyperplasia, whereby smoothmuscle cell and myofibroblast proliferation and extracellular matrixaccumulation cause thickening of the intima in the graft and in theadjoining vessels, and ultimately lead to failure of the graft; and, (2)occlusion of the graft, whereby platelet adhesion and activation at thelumenal surface of the graft initiates thrombosis which, particularly insmaller bore vessel grafts, typically leads to complete occlusion of thegraft.

Research over the past several decades has yet to produce a synthetic orbiosynthetic small bore vascular graft which can approach the patencyrates of autologous vessels. Since small bore grafts have a highersurface area to volume ratio and lower flow rates than larger grafts,the interaction of the graft with the blood is much greater. Plateletadhesion and activation at the lumenal surface of the graft are muchmore likely to result in complete graft occlusion. Larger vasculargrafts are able to remain patent despite a layer of clot lining thelumen because this layer of clot undergoes constant remodeling andessentially maintains a constant thickness. In contrast, clotting on thesurface of a graft smaller than 6 mm in inner diameter has a snowballeffect and results in a continuous growth of the surface clot until theentire graft is occluded.

Currently, non-synthetic or biological small bore grafts are routinelyused as an arterial replacement since nothing has proven to performnearly as well as the autologous saphenous vein or internal mammaryartery, which are the conventional biological materials used as a smalldiameter vascular graft. The use of these vessels requires additionalsurgery, particularly in the case of the saphenous vein, whereby theentire length of the leg must be opened to remove the vessel. Theharvesting surgery increases the total operating time and can also leadto complications and discomfort. Furthermore, a small percentage ofpatients do not have autologous vessels suitable for, harvesting. Insome cases, the vessels are not available due to previous surgery, whilein other cases, the vessel may be too small or varicose.

Even larger bore vessel and organ prosthetic grafts, however, sufferfrom complications associated with smooth muscle proliferation,compliance mismatch with native vessels, and poor endothelialization dueto blood shear stresses and mechanical damage. Therefore, researchershave focused much effort on the development of bioinert andhemocompatible graft materials. However, a completely non-foulingsurface has yet to be discovered and many now view the quest for such amaterial as unrealistic.

Rather than creating a non-fouling surface, others have focused onrecreating the natural blood contacting interface in the body by seedingvascular grafts with endothelial cells (See for example, U.S. Pat. No.5,723,324 to Bowlin et al.; U.S. Pat. No. 5,674,722 to Mulligan et al.,U.S. Pat. No. 5,785,965 to Pratt et al., U.S. Pat. No. 5,766,584 toEdelman et al.). Although a small number of grafts seeded lumenally withendothelial cells have been implanted clinically outside of the UnitedStates, and improved patencies over non-seeded grafts have beenobserved, this approach has generally enjoyed mixed success, and theconcept still faces many challenges. First, it is necessary that thecells used to seed the graft be autologous or otherwise non-immunogenicto avoid recognition and destruction of the cells by the patient'simmune system. To obtain autologous endothelial cells from a patient,the cells must be harvested from an isolated blood vessel. Theharvesting surgical procedure not only increases prosthetic implantpreparation time, but can also lead to complications and discomfort forthe patient.

Second, retention of the cells on the graft surface after implantationhas been an issue. A number of methods have been disclosed to addressthis issue, and include forcible injection of endothelial cells into thegraft, preclotting and seeding the lumenal surface of the graft, staticadhesion-seeding of the lumen, vacuum seeding of the lumen, seeding thelumen in an extracellular matrix, and seeding of the lumen usingelectrostatic and gravitational forces. These methods are reviewed ordisclosed in more detail in U.S. Pat. No. 5,723,324, ibid. Additionally,it has been suggested that flow conditioning the seeded graft in vitroprior to implantation would improve cell retention by allowing the cellsto secrete adhesion factors in response to slowly increasing shear rates(Dardik, et al., 1999, J Vasc Surg 29: 157-67; Ballerman et al., 1995,Blood Purif 13: 125-34; and Ott and Ballerman, 1995, Surgery 117:334-9). Although there is some evidence that methods such asconditioning may improve cell retention, all of these methods add yetanother level of complexity to the seeding process and it is still notclear that significantly improved cellular retention can be achieved.

Therefore, there is a need for prosthetic grafts for use in the repairand replacement of vessels and organs in contact with blood flow thathave improved long term patency and success rates, and which reduce thestress and discomfort experienced by the patient.

SUMMARY OF THE INVENTION

One embodiment of the present invention relates to a prosthetic graftfor containment of blood flow in vivo. The graft includes: (a) a porousprosthetic implant for containing blood in vivo, the prosthetic implanthaving an outer surface that is not in contact with blood flow in vivoand an inner surface that is in contact with blood flow in vivo, theinner surface defining an interior space for containment of blood flow;and, (b) adherent cells adhered to the outer surface of the porousprosthetic implant. The adherent cells are transfected with at least onerecombinant nucleic acid molecule operatively linked to a transcriptioncontrol sequence, the recombinant nucleic acid molecule encoding aprotein that enhances patency of said prosthetic implant.

The prosthetic implant can be configured as any blood containing vesselincluding, but not limited to, a prosthetic vessel, an artificial heart,a left ventricle assist device and/or a dialysis shunt. Prostheticvessels include small, medium and large bore prosthetic vessels. Suchvessels include venous and arterial prosthetic vessels. The implant canbe constructed of any biological or non-biological material whichincludes, but is not limited to, highly resilient polyester, expandedpolytetrafluorethylene (ePTFE), high porosity ePTFE, non-immunogenicxenogeneic tissue, porous silicon rubber, porous polyurethane, porousdegradable polymer, and/or porous copolymers. In preferred embodiments,the prosthetic implant is non-immunogenic, non-calcific, and/or has apore size of from about 0.1 μm to about 500 μm, and more preferably,from about 0.2 μm to about 100 μm.

The adherent cells can be any adherent cells and include, but are notlimited to, fibroblasts, mesenchymal stem cells, bone marrow stem cells,embryonal stem cells, adipocytes, keratinocytes, vascular smooth musclecells, platelets, and cells which have been genetically engineered to beadherent. In one embodiment, the cells are fibroblasts. The cells aretransfected with at least one recombinant nucleic acid molecule encodingat least one protein that enhances patency of the prosthetic implant.Such proteins can include, but are not limited to, a protein thatenhances angiogenesis in the vascular bed downstream of the prostheticgraft, a protein that enhances angiogenesis transmurally and into theinterior space of the prosthetic implant to endothelialize the innersurface of the prosthetic implant, a protein that inhibits thrombosis, aprotein that causes thrombolysis, a protein that inhibits smooth musclemigration and/or proliferation, and a vasodilator protein. Specificexamples of such proteins are described in detail below.

In one embodiment of the present invention, the proteins are expressedby the adherent cells ex vivo, and secreted by the cells ex vivo and/orin vivo. In another embodiment, the proteins are expressed and secretedby the adherent cells in vivo. Preferably, the proteins are secretedinto the pores of the implant and perfuse through the pores and into theinner surface of the implant. In one embodiment, the transcriptioncontrol sequence includes an inducible promoter, so that the expressionof the protein can be up- and/or down-regulated ex vivo or in vivo. Suchan inducible promoter can be regulated, for example, by a compound thatinduces the promoter, including, but not limited to, an antibiotic, ahormone, a transcription factor and/or by a treatment such as internalor external radiation (e.g., X-ray).

Another embodiment of the present invention relates to a vascular graft,which includes: (a) a porous prosthetic vessel having a perivascularsurface and a lumenal surface; and (b) adherent cells adhered to theperivascular surface of the porous prosthetic vessel. The adherent cellsare transfected with at least one recombinant nucleic acid moleculeoperatively linked to a transcription control sequence, the recombinantnucleic acid molecule encoding a protein that enhances patency of theprosthetic vessel.

Yet another embodiment of the invention relates to a prosthetic graftfor containment of blood flow in vivo which includes: (a) a porousprosthetic implant for containing blood in vivo, having an outer surfacethat is not in contact with blood flow in vivo and an inner surface thatis in contact with blood flow in vivo, whereby the inner surface definesan interior space for containment of blood flow; and, (b) adherent cellsadhered to the outer surface of the porous prosthetic implant. In thisembodiment, the adherent cells express and secrete a protein thatenhances patency of the prosthetic implant. In one aspect of thisembodiment of the that have been genetically modified to be adherent.

Yet another embodiment of the present invention relates to a method forproducing a prosthetic graft, which includes the step of applyingadherent cells to a porous prosthetic implant for containing blood invivo, wherein the prosthetic implant has an outer surface and an innersurface that defines an interior space for containment of blood flow.The adherent cells are applied to the outer surface of the prostheticimplant. The adherent cells are transformed with at least onerecombinant nucleic acid molecule operatively linked to a transcriptioncontrol sequence, the recombinant nucleic acid molecule encoding aprotein that enhances patency of the prosthetic implant. Othercharacteristics of the prosthetic implant are described above. Such amethod preferably enhances naturally occurring endothelialization of theinner surface of the implant, inhibits thrombosis in the implant,inhibits thrombosis of the inner surface of the prosthetic implant dueto smooth muscle migration and/or proliferation in the implant, and/orenhances formation of a neointima in the inner surface of the implant.

The step of applying can be performed by any method, including by aprogrammable mechanical graft rotator. When the implant is a prostheticvessel, the step of applying includes seeding the outer surface of thevessel uniformly in both radial and longitudinal directions on thevessel. In one embodiment, the graft is incubated after the step ofapplying for about 5 minutes to about 14 days.

Another embodiment of the present invention relates to a method ofimplantation of a prosthetic graft for containment of blood flow. Such amethod includes the step of implanting a prosthetic graft as describedabove into a patient. In one embodiment, the adherent cells areautologous to the patient. In another embodiment, the adherent cells arefrom a cell selected from the group of undifferentiated stem cell linesand/or embryonal cell lines.

If the transcription control sequence operatively linked to the at leastone recombinant nucleic acid molecule includes an inducible promoter,the cells can be induced to express the protein either in vitro, priorto implantation of the graft into a patient, or in vivo, afterimplantation of the graft into a patient. Other characteristics of sucha graft are described above.

Yet another embodiment of the present invention relates to a method forimplantation of a prosthetic graft for containing blood flow in apatient. Such a method includes the steps of: (a) harvesting fibroblastcells from a patient in need of a prosthetic graft for containing bloodflow; (b) transfecting the fibroblast cells with an isolated nucleicacid molecule encoding a protein that enhances patency of the graft; (c)applying the transfected fibroblast cells onto a surface of a prostheticimplant configured for containing blood flow in vivo for a timesufficient to allow the fibroblast cells to adhere to the surface toform a prosthetic graft, wherein the surface is not in contact withblood flow in vivo; and, (d) implanting the prosthetic graft into thepatient.

Another embodiment of the present invention relates to a method ofenhancing endothelialization of a vascular graft. Such a method includesthe step of applying adherent cells to a porous prosthetic vessel havinga perivascular surface and a lumenal surface, wherein the adherent cellsare adhered to the perivascular surface of the prosthetic vessel. Theadherent cells are transfected with at least one recombinant nucleicacid molecule operatively linked to a transcription control sequence,the recombinant nucleic acid molecule encoding a protein that enhancesendothelialization of the prosthetic vessel. The protein is expressedand secreted by the adherent cells and perfuses through pores in theprosthetic vessel to the lumenal surface of the prosthetic vessel toenhance endothelialization of the graft at the inner surface.

BRIEF DESCRIPTION OF THE FIGURES OF THE INVENTION

FIG. 1A is a bar graph showing the release of VEGF under staticconditions by a prosthetic graft of the present invention.

FIG. 1B is a bar graph showing the release of VEGF under staticconditions by a prosthetic graft of the present invention.

FIG. 1C is a bar graph showing the release of VEGF under staticconditions by a prosthetic graft of the present invention.

FIG. 1D is a bar graph showing the average release of VEGF under staticconditions according to FIGS. 1A-1C by a prosthetic graft of the presentinvention.

FIG. 2A is a bar graph showing the release of VEGF under shear stress of1.5 dyn/cm² by a prosthetic graft of the present invention.

FIG. 2B is a bar graph showing the release of VEGF under shear stress of1.5 dyn/cm² by a prosthetic graft of the present invention.

FIG. 2C is a bar graph showing the release of VEGF under shear stress of1.5 dyn/cm² by a prosthetic graft of the present invention.

FIG. 2D is a bar graph showing the average release of VEGF under shearstress of 1.5 dyn/cm² according to FIGS. 2A-2C by a prosthetic graft ofthe present invention.

FIG. 3A is a bar graph showing the release of VEGF under shear stress of10 dyn/cm² by a prosthetic graft of the present invention.

FIG. 3B is a bar graph showing the release of VEGF under shear stress of10 dyn/cm² by a prosthetic graft of the present invention.

FIG. 3C is a bar graph showing the release of VEGF under shear stress of10 dyn/cm² by a prosthetic graft of the present invention.

FIG. 3D is a bar graph showing the average release of VEGF under shearstress of 10 dyn/cm² according to FIGS. 3A-3C by a prosthetic graft ofthe present invention.

FIG. 4 is a bar graph showing production of VEGF in vivo by a prostheticgraft of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to an improved prosthetic graftand methods of making and using such a graft, for the bypass,replacement or repair of vessels and organs that are in contact withblood flow. The prosthetic graft of the present invention has along-term patency and success rate that is superior to other previouslydescribed prosthetic grafts designed for such use.

More particularly, one embodiment of the present invention relates to aprosthetic graft for containment of blood flow in vivo. The prostheticgraft comprises a porous prosthetic implant for containing blood invivo, wherein the prosthetic implant has an outer surface that is not incontact with blood flow in vivo and an inner surface that is in contactwith blood flow in vivo. The inner surface defines an interior space forcontainment of blood flow. The prosthetic graft additionally includesadherent cells that are adhered to the outer surface of the porousprosthetic implant. The adherent cells are transfected with at least onerecombinant nucleic acid molecule operatively linked to a transcriptioncontrol sequence, and each recombinant nucleic acid molecule encodes oneor more proteins that enhance patency of the prosthetic implant.

The present inventors have discovered that prosthetic implants of thepresent invention have several advantages over other prosthetic graftsof the same type which are currently known in the art.

First, the use of adherent cells which recombinantly produce one or moreproteins that enhance patency of the prosthetic implant eliminates theneed to seed the lumenal surface of the graft with endothelial cells orwith proteins to enhance the endothelialization and neovascularizationof the graft. Prior to the present invention, considerable researcheffort has been expended to determine suitable means of seeding graftswith endothelial cells to enhance the natural neovascularizationprocesses. Other groups have provided selected proteins directly to thegraft lumenal or extralumenal surface for similar reasons. When proteinsare directly provided to the graft, special care must be taken to ensuresterility of the proteins. Efforts to sterilize the proteins and deliverand maintain the proteins at the graft for a time sufficient to providea therapeutic benefit introduces problems associated with degradation ofthe proteins and/or loss of protein activity.

In contrast, in the prosthetic graft of the present invention, theadherent recombinant cells express and secrete one or more proteinswhich diffuse through the porous prosthetic implant to the lumenalsurface of the graft, whereby the proteins are effective to enhancepatency, including inducing natural neovascularization and particularly,endothelialization, of the graft, as well as inhibiting processes whichwould cause occlusion and failure of the graft (i.e., thrombosis, smoothmuscle cell migration and proliferation, platelet adhesion andactivation, extracellular matrix accumulation). Cell culture and graftseeding can easily be accomplished under sterile conditions ex vivo, andthe need to sterilize individual proteins is eliminated. In addition,problems related to degradation and loss of protein activity areeliminated by using recombinant cells to express the protein at thesite. The use of recombinant cells to express the desired proteinadditionally results in the use of smaller amounts of proteins andincreased graft success. Additionally, the recombinant cells can beengineered to have inducible expression of the proteins, so that proteinexpression can be turned on and off in vivo as necessary.

Second, seeding the prosthetic implant on an outer surface of theimplant (e.g., the perivascular surface) where the graft is not indirect contact with blood flow eliminates the complications associatedwith denudation due to blood shear stresses on cells which are seeded onthe inner, or lumenal surface of such implants. As discussed in theBackground section above, researchers have focused intently on a varietyof methods for seeding vascular grafts with endothelial cells to enhancethe rate of graft healing and endothelialization. The prosthetic graftof the present invention eliminates the need to expend the considerabletime and effort on interlumenal endothelial seeding and/or other complexprocesses associated with the use of endothelial cells, which caninvolve extensive manipulation of the graft and in many cases, stillmeets with only limited success.

Third, the use of the recombinant adherent cells according to thepresent invention not only eliminates the need to seed the lumenalsurface of the implant with proteins or endothelial cells, but theadherence of the cells to the outer surface of the implant allows thegraft to be easily manipulated ex vivo and in vivo, without the need forthe use of additional devices or other extraneous binding compounds tocontain the cells at the graft site. In a non-graft medical procedurefor the repair of damaged physiological tubular structures, it has beenproposed to introduce proteins or cells at an extralumenal site adjacentto a lesion in the damaged physiological tubular structure for diffusionof proteins into the damaged tissue (See for example, U.S. Pat. No.5,540,928 to Edelman et al., U.S. Pat. No. 5,455,039 to Edelman et al.,U.S. Pat. No. 5,527,532 to Edelman et al., U.S. Pat. No. 5,766,584 toEdelman). Such methods, however, require the use of additional matrices,devices, sheaths and/or binding agents to deliver and maintain the cellsor proteins at the extralumenal surface of the damaged vessel to berepaired, and are therefore undesirable for use in the prosthetic graftof the present invention. Additionally, devices such as controlledrelease or wicking devices are required to provide a source by whichsuch proteins can be continuously administered to the graft over aperiod of time sufficient to obtain a biological effect at the lumenalsurface of the graft, adding yet another level of complexity to thedelivery of proteins.

In contrast, the prosthetic graft of the present invention isessentially self-sufficient in that once the graft is prepared andimplanted, there is no need for additional manipulation of the graft orrenewed provision of cells or proteins at the graft site. The prostheticgraft of the present invention can therefore perform over extendedperiods of time without further surgical intervention. In addition,adherent cells suitable for use in the graft of the present inventioncan be transfected and cultured, the protein expression can be verified,the protein expression levels can be adjusted, and the cells can beapplied and cultured on the prosthetic graft, with all such steps beingaccomplished ex vivo, prior to implantation of the graft into the host.

Fourth, the adherent cells useful in the present prosthetic graft aresuperior to the relatively non-adherent endothelial cells which havepreviously been used to seed vascular grafts, including recombinantendothelial cells. More particularly, suitable adherent cells for use inthe present invention, which can include fibroblasts, mesenchymal stemcells, bone marrow stem cells (e.g., undifferentiated stem cells fromadult bone marrow), embryonal stem cells, adipocytes, keratinocytes,vascular smooth muscle cells, platelets, and other cells which have beengenetically engineered to be adherent, are all relatively easy to obtainas autologous cells from the patient in need of the graft and/or arerelatively non-immunogenic (i.e., embryonal stem cells). For example,fibroblasts and keratinocytes can be obtained through non-invasiveharvesting methods such as a skin biopsy punch, and platelets can beobtained through a simple blood draw. Adipocytes can be obtained byrelatively non-invasive skin biopsy or liposuction. In contrast,endothelial cells must be harvested from an isolated blood vessel. Asdiscussed above, obtaining a blood vessel from a patient requiresadditional surgery and can also lead to complications and discomfort forthe patient. Additionally, embryonal and mesenchymal stem cells, whichcan be maintained as recombinant cells lines, are relativelynon-immunogenic and therefore would eliminate the need to use autologouscells in the graft.

Moreover, suitable adherent cells for use in the present invention, andparticularly fibroblast cells, are a much heartier cell type thanendothelial cells and they grow and maintain their phenotype much betterthan endothelial cells in vitro. In particular, fibroblasts arerelatively innocuous in terms of endogenous protein expression, andtherefore, they make ideal host cells for the expression of heterologousproteins. Finally, embryonal stem cells, bone marrow stem cells andmesenchymal stem cells have the additional advantage that such cells canbe induced to differentiate into a desired cell type and therebyendogenously produce certain proteins by addition of exogenous growthfactors.

According to the present invention, a “prosthetic graft” is defined asan artificial substitute organ or tissue used forimplantation/transplantation, and in the present invention, theprosthetic graft is used for the bypass, replacement or repair of anorgan or tissue that contains blood flow in vivo. A prosthetic graft ofthe present invention includes at least two components: (1) a porousprosthetic implant for containing blood in vivo; and, (2) adherent cellsadhered to an outer surface of the implant, whereby the cells aretransfected with at least one recombinant nucleic acid molecule thatencodes one or more proteins that enhance patency of the implant.Therefore, reference herein to the prosthetic graft is intended toencompass both the prosthetic implant itself and the adherent cells, andreference to the prosthetic implant is intended to reference only theportion of the graft which is the structural prosthesis for containmentof blood flow. As used herein, to “contain blood flow” or “configuredfor containment of F blood flow” means that the implant/portion of thegraft is configured to be a vessel, chamber, or other such structurethat has or partially defines an interior space that confines anddirects the flow of blood into, through and out of the graft. In apreferred embodiment, the prosthetic graft of the present invention isused to repair, replace or bypass a blood vessel, a heart, a chamber ofa heart, or is used for vascular access, such as a dialysis shunt. Theprosthetic implant can therefore include, but is not limited to, aprosthetic vessel, an artificial heart, a left ventricle assist device,or a dialysis shunt.

As discussed above, the prosthetic implant has an outer surface and aninner surface. The outer surface (e.g., external surface) is definedherein as a surface of the implant which is not in contact with bloodflow. In a prosthetic vessel implant, the outer surface is also referredto as the perivascular surface of the vessel implant. The inner surface(e.g., internal surface) is defined herein as a surface of the implantwhich is in contact with blood flow. The inner surface of a prostheticvessel implant can also be referred to as the lumenal surface. The innersurface defines an interior space enclosed by the implant through whichblood can flow. The inner surface, or lumenal surface, of the implant isthe surface upon which neovascularization events, such asendothelialization of the graft, and other events related to improvedpatency, such as inhibition of smooth muscle proliferation, occur.

According to the present invention, improved or enhanced patency isdefined to encompass enhancement (i.e., increase, improvement) of any ofthe biological processes that contribute to the initiation, development,and/or maintenance of neovascularization of a blood-containing vessel ororgan, as well as inhibition (i.e., decrease, diminution) of any of thebiological processes that contribute to occlusion (i.e., closing,blocking off, obstruction of a vessel or organ), intimal hyperplasia(i.e., an increase in thickness of intimal tissue due to an increase inthe number of its constituent cells) and/or failure of ablood-containing vessel or organ. As such, the term “enhanced patency”or “improved patency” can be used to generally refer to events whichinclude: enhanced angiogenesis (i.e., blood vessel formation) in thevascular bed downstream of the prosthetic graft, enhanced angiogenesistransmurally and into the interior space of the prosthetic implant toendothelialize (i.e., attract, grow and establish endothelial cells) theinner surface of the prosthetic implant, inhibition of thrombosis, andinhibition of smooth muscle migration and/or proliferation.

According to the present invention, neovascularization includes any ofthe biological processes involved in the development and maintenance ofa natural vessel or organ through which blood flows. More particularly,neovascularization results in the attraction, growth and establishmentwithin the porous prosthetic implant of cells and tissue that aresubstantially similar to the natural tissue that defines blood vesselsand/or the walls of a heart, for example. By “substantially similar”tissue, it is intended that the new cells and tissue formed on andwithin the implant be similar enough in composition, cellular/tissueorganization and function to the naturally occurring tissue which isbeing repaired, replaced or bypassed, that the new tissue is capable ofeffectively performing the functions of the naturally occurring tissuein the graft recipient under normal physiological conditions. Inparticular, formation of a substantially natural new intimal surface(i.e., a neointima) is desirable in the prosthetic graft. A naturalintimal surface primarily comprises a uniform covering of endothelialcells over which blood flows. Under normal physiological conditions,blood flows over such a surface without forming thromboses, sinceendothelial cells have natural characteristics which inhibit thrombosis.Preferably, enhanced neovascularization of a prosthetic graft of thepresent invention includes one or more of the following processes:angiogenesis (i.e., blood vessel formation) in the vascular beddownstream of the prosthetic graft and angiogenesis transmurally andinto the interior space of the prosthetic implant to endothelialize(i.e., attract, grow and establish endothelial cells) the inner surfaceof the prosthetic implant.

As used herein, the ability of a protein to “enhance neovascularization”refers to the ability of the protein to initiate, regulate, provideand/or contribute to any process involved in successfulneovascularization as described above, as well as the ability to enhance(i.e., increase, upregulate) any naturally occurring neovascularizationwhich is occurring in the absence of the protein.

Preferably, inhibition of: occlusion, intimal hyperplasia and/or failureof a graft includes one or more of the following processes: inhibitionof thrombosis, enhancement of thrombolysis, and/or inhibition of smoothmuscle migration and/or proliferation.

The prosthetic implant of the present invention is porous to allow theprotein(s) that are expressed and secreted by the adherent cells toperfuse into and through the implant from the outer surface of theimplant to the inner surface of the implant, where inhibition of eventsthat decrease patency and enhancement of events that increase patency(e.g., neovascularization of the implant, including endothelializationof the inner surface of the implant), can occur. In this fashion, theprosthetic implant of the present invention effectively serves, in part,as a delivery system for biological compounds. The size of the pores ofthe implant can be any minimal size which allows the passage of proteinstherethrough, and any maximal size which maintains the mechanicalproperties of the graft (e.g., support, configuration). Preferably, thesize of the pores of the implant are from about 0.1 μm to about 50 μm,and more preferably, from about 0.2 μm to about 100 μm.

The porous prosthetic implant for use in the prosthetic graft of thepresent invention can be constructed of any material that is porous andsuitable for use in a prosthesis for the containment of blood flow invivo. The material(s) can be biological (i.e., natural), synthetic, orcombinations thereof. Preferably, the material(s) are non-immunogenicand/or non-calcific. Such materials include, but are not limited to,highly resilient polyester, expanded polytetrafluorethylene (ePTFE),high porosity ePTFE, non-immunogenic xenogeneic tissue (e.g.,dye-mediated, cross-linked collagen), porous silicon rubber, porouspolyurethane, porous degradable polymer and/or porous copolymers.

In one embodiment of the present invention, a prosthetic implantsuitable for use in a prosthetic graft of the present invention is asmall bore prosthetic vessel. The present invention is particularlyvaluable for use in replacement and repair of small bore vessels,because the graft eliminates problems with occlusion and hyperplasiawhich have previously made the use of non-autologous and non-biologicalsmall bore grafts difficult if not impossible. A small bore graft whichdoes not require the harvesting of autologous vessels would be a lesstraumatic option for the patient and also provide an option for thosepatients with no suitable vessels. According to the present invention, asmall bore graft is defined as a vessel with an inner diameter of lessthan about 6 mm. The present invention is also useful for prostheticimplant that is a medium or large bore vessel. A medium bore vessel isdefined herein as a vessel with an inner diameter of from about 6 mm toabout 12 mm, and a large bore vessel is defined herein as a vessel withan inner diameter of from about 12 mm to about 38 mm. A prostheticvessel implant of the present invention can be used for any suitablepurpose, including arterial or venous replacement, or as a dialysisshunt.

Accordingly, in one embodiment of the present invention, the prostheticgraft is a vascular graft which includes: (a) a porous prosthetic vesselhaving a perivascular surface and a lumenal surface; and (b) adherentcells adhered to the perivascular surface of the porous prostheticvessel, wherein the adherent cells are transfected with a recombinantnucleic acid molecule operatively linked to a transcription controlsequence. The recombinant nucleic acid molecule encodes a protein thatenhances patency of the prosthetic vessel.

The prosthetic graft of the present invention also includes adherentcells adhered to the outer surface of the prosthetic implant. Theadherent cells are transfected with at least one recombinant nucleicacid molecule operatively linked to a transcription control sequence.Each recombinant nucleic acid molecule encodes one or more proteins thatenhance patency of the prosthetic implant.

According to the present invention, an adherent cell is any cell thatnaturally adheres to a surface in the absence of factors or conditionswhich inhibit the adhesive properties of the cell (i.e., under normalconditions), or that can be induced to be adherent. More particularly,an adherent cell is characterized herein as a cell that is capable ofadhering to a surface of a prosthetic implant as described herein underin vitro and/or in vivo culture conditions that are suitable for thenormal growth and propagation of the given cell type, such adherencebeing capable of occurring without the assistance of additionalexogenous “binding factors” (e.g., by polymeric compositions, collagen,fibronectin, fibrin, attachment peptides, laminin, etc.) to bind thecell to the surface (i.e., the adherence ability is a naturalcharacteristic of the cell type). Such a naturally adherent cell canhave the characteristic of being naturally non-adherent or adherentdepending upon induction of the characteristic or upon placement of thecell in an appropriate environment. A suitable adherent cell for use inthe present invention is capable of naturally adhering to the surface ofan implant as described above, and is further capable of remainingadhered to the surface of the implant during normal in vitro culturemanipulations, implantation procedures, and normal in vivo stresses thatoccur at the outer surface of the graft. Typically, at least about 60%,and preferably at least about 70%, and more preferably, at least about80%, and even more preferably, at least about 90% of the total number ofadherent cells that are initially adhered to an implant outer surfaceremain adhered to the surface during such manipulations and procedures.Cells that have such characteristics are known in the art and include,but are not limited to, fibroblasts, mesenchymal stem cells, bone marrowstem cells, embryonal stem cells, adipocytes, keratinocytes, vascularsmooth muscle cells, and platelets.

According to the present invention, an adherent cell can also include acell that has been genetically modified to be adherent, such theadherency becomes a natural characteristic of the genetically modifiedcell. A genetically modified cell is a cell that has been modified(i.e., mutated or changed) within its genome and/or by recombinanttechnology (i.e., genetic engineering) from its normal (i.e., wild-typeor naturally occurring) form. For example, an endothelial cell is notconsidered to be an adherent cell according to the present invention,unless such cell has been genetically modified to be more adherent thana naturally occurring endothelial cell, in which case such a geneticallymodified endothelial cell is encompassed by the present invention. Asdiscussed above, one advantage of the present invention over previouslydescribed prosthetic grafts is that the use of adherent cells on theouter surface of the graft eliminates the need for additional devices,delivery vehicles or binders that complicate preparation andcompatibility of the graft and which can compromise the viability andstability of the recombinant cells and proteins produced by the cells.

Preferred adherent cells for use in the present invention include, butare not limited to fibroblasts, mesenchymal stem cells, bone marrow stemcells, embryonal stem cells, adipocytes, keratinocytes, vascular smoothmuscle cells, platelets, and cells which have been geneticallyengineered to be adherent, with fibroblasts being particularlypreferred.

The adherent cells of the present invention are transfected with atleast one recombinant nucleic acid molecule that encodes one or moreproteins that enhance patency in the prosthetic implant. Enhancedpatency has been previously defined herein. According to the presentinvention, proteins that are particularly useful in enhancing patency inthe prosthetic graft of the present invention include: a protein thatenhances angiogenesis in the vascular bed downstream of the prostheticgraft, a protein that enhances angiogenesis transmurally and into theinterior space of the prosthetic implant to endothelialize the innersurface of the prosthetic implant, a protein that inhibits thrombosis, aprotein that causes thrombolysis, a protein that inhibits smooth musclemigration and/or proliferation, and/or a vasodilator protein.

Examples of proteins which are angiogenic (i.e., enhance or initiateangiogenesis) and/or are useful growth factors for enhancing patencyinclude, but are not limited to: vascular endothelial growth factor(VEGF), platelet-induced growth factor (PIGF), transforming growthfactor μ1 (TGFβ1), acidic fibroblast growth factor (aFGF), basicfibroblast growth factor (bFGF), transforming growth factor α (TGFα),epidermal growth factor, osteonectin, angiopoietin 1 (Ang1), Ang2,insulin-like growth factor (IGF), platelet-derived growth factor AA(PDGF-AA), PDGF-AB and PDGF-BB. Examples of proteins which are usefulfor inhibiting thrombosis and/or causing thrombolysis include, but arenot limited to: tissue plasminogen activator (TPA), streptokinase,hirudin V, αv-βIII, and urokinase plasminogen activator (uPA). Anexample of a protein which is useful for inhibiting smooth muscle cellmigration and/or proliferation includes, but is not limited to nitricoxide synthase (NO synthase). An example of a vasodilator proteinincludes, but is not limited to prostacyclin. Other suitable proteinswhich can perform the above-described functions or otherwise enhancepatency will be known to those of skill in the art. In addition, theamino acid and nucleic acid sequences for these proteins are known andtherefore, the proteins can be readily produced recombinantly by a hostcell using recombinant technology that is well known in the art.

According to the present invention, in one embodiment, all of theadherent cells which are adhered to the outer surface of the prostheticimplant can be transfected with the same recombinant nucleic acidmolecule(s), so that each cell expresses the same recombinantprotein(s). In another embodiment, adherent cells expressing differentrecombinant nucleic acid molecule(s) can be combined and adhered to thesame implant. As such, several different proteins can be expressed onthe same implant, and the proportions of the various proteins can becontrolled by the proportion of adherent cells expressing each proteinthat are adhered to the outer surface of the implant or by the level ofexpression of the respective proteins.

Similarly, a single adherent cell is transfected with at least onerecombinant nucleic acid molecule, but it is within the scope of thepresent invention that a single adherent cell can be transformed withtwo or more different recombinant nucleic acid molecules, so that asingle adherent cell can express one, two, or multiple recombinantproteins. A single adherent cell can also be transfected with a singlerecombinant nucleic acid molecule that expresses one, two or multipleproteins, which can be under the control of the same transcriptioncontrol sequence, or under the control of different transcriptioncontrol sequences. In the case of expression of two or more recombinantnucleic acid molecules, the expression levels of the different moleculescan be independently regulated, by, for example, controlling the copynumber of the different recombinant nucleic acid molecules or by usingdifferent promoters to express the different proteins. According to thepresent invention, reference to “one” or “a single” recombinant nucleicacid molecule is intended to refer to one type of molecule or oneparticular sequence, but it is not to be interpreted to mean that asingle host cell contains only one copy number of the molecule. Forexample, a host cell that is transfected with a single recombinantnucleic acid molecule can have and express one or multiple copies of thesame recombinant nucleic acid molecule. It is to be noted that the term“a” or “an” entity generally refers to one or more of that entity; forexample, a protein refers to one or more proteins, or to at least oneprotein. As such, the terms “a” (or “an”), “one or more” and “at leastone” can be used interchangeably herein. It is also to be noted that theterms “comprising”, “including”, and “having” can be usedinterchangeably.

An adherent cell suitable for use in the present invention is producedby transforming a host adherent cell with at least one recombinantnucleic acid molecule, each comprising one or more isolated nucleic acidsequences encoding a protein as described above and operatively linkedto one or more transcription control sequences. The transcriptioncontrol sequence(s) are typically contained within an expression vector.An expression vector is a DNA or RNA vector that is capable oftransforming a host cell and of effecting expression of a specifiednucleic acid molecule. Preferably, the expression vector is also capableof replicating within the host cell. In the present invention,expression vectors are typically plasmids, although any other expressionvectors, such as retroviral vectors, are encompassed by the presentinvention. Expression vectors of the present invention include anyvectors that function (i.e., direct gene expression) in an adherent hostcell. Such a vector can contain nucleic acid sequences that are notnaturally found adjacent to the isolated nucleic acid molecules to beinserted into the vector. The vectors can be used in the cloning,sequencing, and/or otherwise manipulating of nucleic acid molecules.

The phrase “operatively linked” refers to linking a nucleic acidmolecule to a transcription control sequence in a manner such that themolecule is able to be expressed when transfected (i.e., transformed,transduced or transfected) into a host cell. Transcription controlsequences are sequences which control the initiation, elongation, andtermination of transcription. Particularly important transcriptioncontrol sequences are those which control transcription initiation, suchas promoter, enhancer, operator and repressor sequences. Suitabletranscription control sequences include any transcription controlsequence that can function in at least one of the recombinant cellsuseful in the prosthetic graft and method of the present invention. Avariety of such transcription control sequences are known to thoseskilled in the art. Preferred transcription control sequences includethose which function in mammalian cells and include mammalian andretroviral transcription control sequences. Even more preferredtranscription control sequences include those which function in a celltype selected from the group consisting of fibroblasts, mesenchymal stemcells, bone marrow stem cells, embryonal stem cells, adipocytes,keratinocytes, vascular smooth muscle cells, and/or platelets.

In accordance with the present invention, an isolated nucleic acidmolecule is a nucleic acid molecule that has been removed from itsnatural milieu (i.e., that has been subject to human manipulation). Assuch, “isolated” does not reflect the extent to which the nucleic acidmolecule has been purified. An isolated nucleic acid molecule caninclude DNA, RNA, or derivatives of either DNA or RNA. There is nolimit, other than a practical limit, on the maximal size of a nucleicacid molecule in that the nucleic acid molecule can include a portion ofa gene; an entire gene, including regulatory and other untranslatedregions of the gene; multiple genes; or portions thereof.

An isolated nucleic acid molecule useful in the present invention can beobtained from its natural source either as an entire (i.e., complete)gene or a portion thereof capable of forming a stable hybrid with thatgene. An isolated nucleic acid molecule can also be produced usingrecombinant DNA technology (e.g., polymerase chain reaction (PCR)amplification, cloning) or chemical synthesis. Isolated nucleic acidmolecules include natural nucleic acid molecules and homologues thereof,including, but not limited to, natural allelic variants and modifiednucleic acid molecules in which nucleotides have been inserted, deleted,substituted, and/or inverted in such a manner that such modificationsprovide the desired effect within the microorganism.

An allelic variant of a gene having a given nucleic acid sequence is agene that occurs at essentially the same locus (or loci) in the genomeas the gene having the given nucleic acid sequence, but which, due tonatural variations caused by, for example, mutation or recombination,has a similar but not identical sequence. Allelic variants typicallyencode proteins having similar activity to that of the protein encodedby the gene to which they are being compared. Allelic variants can alsocomprise alterations in the 5′ or 3′ untranslated regions of the gene(e.g., in regulatory control regions). Allelic variants are well knownto those skilled in the art.

A nucleic acid molecule homologue can be produced using a number ofmethods known to those skilled in the art (see, for example, Sambrook etal., ibid.). For example, nucleic acid molecules can be modified using avariety of techniques including, but not limited to, classic mutagenesistechniques and recombinant DNA techniques, such as site-directedmutagenesis, chemical treatment of a nucleic acid molecule to inducemutations, restriction enzyme cleavage of a nucleic acid fragment,ligation of nucleic acid fragments, PCR amplification and/or mutagenesisof selected regions of a nucleic acid sequence, synthesis ofoligonucleotide mixtures and ligation of mixture groups to “build” amixture of nucleic acid molecules and combinations thereof. Nucleic acidmolecule homologues can be selected from a mixture of modified nucleicacids by screening for the function of the protein encoded by thenucleic acid and/or by hybridization with a wild-type gene.

Knowing the nucleic acid sequences of certain nucleic acid moleculesencoding proteins useful in the present invention allows one skilled inthe art to, for example, (a) make copies of those nucleic acid moleculesand/or (b) obtain nucleic acid molecules including at least a portion ofsuch nucleic acid molecules (e.g., nucleic acid molecules includingfull-length genes, full-length coding regions, regulatory controlsequences, truncated coding regions). Such nucleic acid molecules can beobtained in a variety of ways including traditional cloning techniquesusing oligonucleotide probes of to screen appropriate libraries or DNAand PCR amplification of appropriate libraries or DNA usingoligonucleotide primers.

Although the phrase “nucleic acid molecule” primarily refers to thephysical nucleic acid molecule and the phrase “nucleic acid sequence”primarily refers to the sequence of nucleotides on the nucleic acidmolecule, the two phrases can be used interchangeably.

It may be appreciated by one skilled in the art that use of recombinantDNA technologies can improve expression of transformed nucleic acidmolecules by manipulating, for example, the number of copies of thenucleic acid molecules within a host cell, the efficiency with whichthose nucleic acid molecules are transcribed, the efficiency with whichthe resultant transcripts are translated, and the efficiency ofpost-translational modifications. Recombinant techniques useful forincreasing the expression of nucleic acid molecules of the presentinvention include, but are not limited to, operatively linking nucleicacid molecules to high-copy number plasmids, integration of the nucleicacid molecules into the host cell chromosome, addition of vectorstability sequences to plasmids, substitutions or modifications oftranscription control signals (e.g. promoters, operators, enhancers)substitutions or modifications of translational control signals,modification of nucleic acid molecules of the present invention tocorrespond to the codon usage of the host cell, and deletion ofsequences that destabilize transcripts. The activity of an expressedrecombinant protein of the present invention may be improved byfragmenting, modifying, or derivatizing nucleic acid molecules encodingsuch a protein.

It is preferred that the nucleic acid molecule encoding proteinaccording to the present invention be cloned under control of anartificial promoter. The promoter can be any suitable promoter that willprovide a level of protein expression required to maintain a sufficientlevel of the protein at the graft site to enhance patency of theimplant, and preferably, to complete neovascularization of the implant.Preferred promoters are inducible promoters, since it is desirable to beable to regulate the expression of the protein ex vivo and/or in vivo atthe site of graft implantation. In one embodiment, the promoter isinducible in vivo, so that the prosthetic graft can be implanted at thedesired site in vivo, and the protein production can be regulated asnecessary to enhance patency. In this embodiment, the protein productioncan be terminated after a time has passed that is sufficient toestablish vascularization and particularly, endothelialization of theimplant, thereby eliminating any undesirable side effects that may becreated by prolonged exposure of the graft recipient to the proteins.Preferably, an, inducible promoter useful in the present invention isinduced by administration of a compound that regulates the promoter tothe graft recipient, such compound being administered in an amount andby a route effective to regulate transcription of the recombinantnucleic acid molecule in the adherent cells. Such a compound caninclude, but is not limited to, an antibiotic, a hormone, or atranscription factor. In another embodiment, instead of a compound, theinducible promoter can be activated by a treatment such as internal orexternal radiation (e.g., X-ray). Alternatively, a promoter may beselected that will be induced upon placement in the in vivo environmentof the implant, by naturally occurring compounds (e.g., hormones) thatenter or are produced by the adherent cells and bind to and induce thepromoter.

The gene dosage (copy number) of a recombinant nucleic acid moleculeaccording to the present invention can also be varied according to therequirements for maximum product formation. In one embodiment, therecombinant nucleic acid molecule encoding a protein useful in thepresent invention is integrated into the chromosome of the host cell.

Proteins expressed by recombinant nucleic acid molecules according tothe present invention are secreted from the cell. Therefore, recombinantnucleic acid molecules used in the present invention typically containsecretory signals (i.e., signal segment nucleic acid sequences) toenable an expressed protein of the present invention to be secreted fromthe cell that produces the protein. Examples of suitable signal segmentsinclude any signal segment capable of directing the secretion of aprotein of the present invention. Preferred signal segments includesignal segments which are naturally associated with the expressedprotein, when such protein is a secreted protein, but can include anysignal segment that functions in a host cell according to the presentinvention.

A protein that enhances patency produced by a recombinant nucleic acidmolecule according to the present invention includes can be afull-length protein (i.e., in its full-length, naturally occurringform), any homologue of such a protein, any fusion protein containingsuch a protein, or any mimetope of such a protein. The amino acidsequences for many patency enhancing proteins disclosed herein, as wellas nucleic acid sequences encoding the same, are known in the art andare publicly available, for example, from sequence databases such asGenBank. Such sequences can therefore be obtained and used to produceproteins and recombinant nucleic acid molecules of the presentinvention.

A homologue is defined as a protein in which amino acids have beendeleted (e.g., a truncated version of the protein, such as a peptide orfragment), inserted, inverted, substituted and/or derivatized (e.g., byglycosylation, phosphorylation, acetylation, myristoylation,prenylation, palmitation, amidation and/or addition ofglycosylphosphatidyl inositol). A homologue of a given protein is aprotein having an amino acid sequence that is sufficiently similar to anaturally occurring protein amino acid sequence that the homologue hassubstantially the same, or enhanced or even reduced biological activitycompared to the corresponding naturally occurring protein.

As used herein, a mimetope (also referred to as a synthetic mimic) of aprotein that enhances patency according to the present invention refersto any compound that is able to mimic the activity of such a protein,often because the mimetope has a structure that mimics the protein.Mimetopes can be, but are not limited to: peptides that have beenmodified to decrease their susceptibility to degradation; anti-idiotypicand/or catalytic antibodies, or fragments thereof; non-proteinaceousimmunogenic portions of an isolated protein (e.g., carbohydratestructures); and synthetic or natural organic molecules, includingnucleic acids. Such mimetopes can be designed using computer-generatedstructures of the naturally occurring protein. Mimetopes can also beobtained by generating random samples of molecules, such asoligonucleotides, peptides or other organic or inorganic molecules, andscreening such samples by affinity chromatography techniques using thecorresponding binding partner.

According to the present invention, a fusion protein is a protein thatincludes a patency-enhancing protein containing domain attached to oneor more fusion segments. Suitable fusion segments for use with thepresent invention include, but are not limited to, segments that can:enhance a protein's stability; enhance the biological activity of apatency-enhancing protein; and/or assist purification of apatency-enhancing protein (e.g., by affinity chromatography). A suitablefusion segment can be a domain of any size that has the desired function(e.g., imparts increased stability, imparts enhanced biological activityto a protein, and/or simplifies purification of a protein). Fusionsegments can be joined to amino and/or carboxyl termini of thepatency-enhancing protein-containing domain of the protein and can besusceptible to cleavage in order to enable straight-forward recovery ofa patency-enhancing protein, if such recovery is desired. Fusionproteins are preferably produced by culturing a recombinant celltransformed with a fusion nucleic acid molecule that encodes a proteinincluding the fusion segment attached to either the carboxyl and/oramino terminal end of a patency-enhancing protein-containing domain.Preferred fusion segments include a metal binding domain (e.g., apoly-histidine segment); an immunoglobulin binding domain (e.g., Proteina; Protein G; T cell; B cell; Fc receptor or complement proteinantibody-binding domains); a sugar binding domain (e.g., a maltosebinding domain); and/or a “tag” domain (e.g., at least a portion ofβ-galactosidase, a strep tag peptide, other domains that can be purifiedusing compounds that bind to the domain, such as monoclonal antibodies).

Suitable host cells to transform include any adherent cell as describedabove that can be transformed with a nucleic acid molecule encoding aprotein useful in the prosthetic graft of the present invention. Hostcells can be either untransformed adherent cells or cells that arealready transformed with at least one nucleic acid molecule (e.g.,nucleic acid molecules encoding one or more proteins useful in thepresent invention). Adherent host cells of the present invention can beendogenously (i.e., naturally) capable of producing the useful proteinsof the present invention in addition to being capable of producing suchproteins after being transformed with at least one recombinant nucleicacid molecule encoding such protein.

Suitable adherent host cells can be obtained from the recipient of theprosthetic graft of the present invention (i.e., autologous cells), froma histocompatible allogeneic donor, from a xenogeneic donor, from anembryonal cell source, and/or from established cell lines that arepropagated in vitro. Methods of obtaining and culturing autologous cellsfrom the graft recipient or allogeneic/xenogeneic cells from a donorvary depending on the type of cell to be obtained, and are well known inthe art. For example, methods for obtaining and culturing fibroblastsand other cells from a donor are described in detail in U.S. Pat. No.5,460,959, to Mulligan et al.; R. Ian Feshney, Culture of Animal Cells,Editor: Wiley-Liss, 3rd ed., 1994; Paw and Zon, 1999, Methods Cell Biol59:39-43; and Hodges-Garcia et al., 1998, In Vitro Cell Dev Biol Anim34(5):364-366; each of which is incorporated herein by reference in itsentirety.

Transformation of a nucleic acid molecule into a cell can beaccomplished by any method by which a nucleic acid molecule can beinserted into the cell. Transformation techniques include, but are notlimited to, transfection, electroporation and microinjection. Methods oftransducing fibroblasts for expression of heterologous proteins aredescribed in detail in U.S. Pat. No. 5,460,959, ibid.; and Ray and Gage,1992, Biotechniques 13(4):598-603, incorporated herein by reference inits entirety.

Proteins which enhance patency are expressed and secreted from adherenthost cells of the present invention by culturing the recombinantadherent cell capable of expressing the protein under conditionseffective to produce the protein. Such conditions include both ex vivoand in vivo conditions. Effective ex vivo culture conditions include,but are not limited to, effective media, bioreactor, temperature, pH andoxygen conditions that permit protein production. An effective mediumrefers to any medium in which a cell is cultured to produce proteinaccording to the present invention. Such medium typically comprises anaqueous medium having assimilable carbon, nitrogen and phosphatesources, and appropriate salts, minerals, metals and other nutrients,such as vitamins. Cells of the present invention can be cultured inconventional fermentation bioreactors, shake flasks, test tubes,microtiter dishes, and petri plates. Culturing can be carried out at atemperature, pH and oxygen content appropriate for a recombinant cell.Such culturing conditions are within the expertise of one of ordinaryskill in the art. An example of suitable culture conditions is describedin the Examples section. Effective in vivo conditions are normalphysiological conditions at the site of the implantation of theprosthetic graft. Additionally, effective in vivo conditions can includethe presence of a promoter inducer, if the transcription controlsequence of the recombinant nucleic acid molecule includes an induciblepromoter.

Prior to, or after adhering an adherent cell of the present invention toa prosthetic implant as described herein, the expression of the desiredrecombinant protein by the recombinant adherent cell can be verified bya method such as, but not limited to, immunoblot or analysis of thebiological activity of the protein to be expressed. If necessary, theexpression level of the protein can be adjusted by methods whichinclude, but are not limited to, recloning the nucleic acid moleculeinto a higher copy number plasmid, integration of the nucleic acidmolecules into the host cell chromosome, addition of vector stabilitysequences to plasmids, substitutions or modifications of transcriptioncontrol signals (e.g., promoters, operators, enhancers), substitutionsor modifications of translational control signals, modification ofnucleic acid molecules to correspond to the codon usage of the hostcell, and deletion of sequences that destabilize transcripts, etc.

Recombinant adherent cells of the present invention are grown in vitroin effective culture Conditions for an amount of time effective toestablish a sufficient number of viable recombinant cells to adhere to aprosthetic implant to be implanted into a patient. The number of cellsthat is sufficient to adhere to a prosthetic implant will vary dependingon the size and shape of the implant, and depending on the amount ofprotein expressed per cell. In general, a prosthetic implant should beseeded on the outer surface with a number of cells sufficient to expressan amount of protein that can diffuse into the graft, and, under in vivoconditions including blood shear forces, within the implant and at theinner surface of the graft, effect the desired biological activity.Determination of these parameters is well within the ability of one ofordinary skill in the art and is described in detail in the Examplessection.

In one embodiment of the method of the present invention, a recombinantnucleic acid molecule encoding a protein that enhances patency asdescribed above is delivered to the outer surface of a prostheticimplant as described herein by a non-cellular delivery vehicle thatadheres to the outer surface of the implant and/or perfuses into thepores of the implant. In this embodiment, instead of using an adherentcell to express and deliver the protein to the implant, a non-cellularvehicle, including, but not limited to: a liposome, an immunoliposome,or a controlled release polymer delivery vehicle, is used to deliver theprotein onto the outer surface of the implant. Upon implantation, theliposome can transfect host cells in the vicinity of the outer surfaceof the graft or which are recruited to the graft with the recombinantnucleic acid molecule, whereby the recombinant nucleic acid molecule isexpressed by the cells.

A liposome delivery vehicle of the present invention comprises a lipidcomposition that is capable of fusing with the plasma membrane of a cellto deliver a nucleic acid molecule or other compound into a cell. Animmunoliposome is a liposome which requires an antibody (conjugated to alipid anchor) not only for specific target cell recognition but also asstabilizer of the otherwise unstable liposome (Ho et al., 1986,Biochemistry 25: 5500-6; Ho et al., 1987a, J Biol Chem 262: 13979-84;and Ho et al., 1987b, J Biol Chem 262: 13973-8; all incorporated hereinby reference in their entireties). Suitable liposomes for use with thepresent invention include any liposome. Preferred liposomes of thepresent invention include those liposomes commonly used in, for example,gene delivery methods known to those of skill in the art. Complexing aliposome with a nucleic acid molecule of the present invention can beachieved using methods standard in the art. A suitable concentration ofa nucleic acid molecule of the present invention to add to a liposomeincludes a concentration effective for delivering a sufficient amount ofnucleic acid molecule into a host cell such that the protein isexpressed at a level sufficient to enhance patency in the graft,including at the inner surface of the graft.

Vehicles for non-cellular delivery of a recombinant nucleic acidmolecule encoding a protein that enhances patency as described abovealso include a controlled release formulation that is capable of slowlyreleasing the recombinant nucleic acid molecule into the graft site. Asused herein, a controlled release formulation comprises a recombinantnucleic acid molecule encoding a protein that enhances patency in acontrolled release vehicle. Suitable controlled release vehiclesinclude, but are not limited to, biocompatible polymers, other polymericmatrices, capsules, microcapsules, microparticles, bolus preparations,osmotic pumps, diffusion devices, liposomes, lipospheres, andtransdermal delivery systems. Other controlled release formulations ofthe present invention include liquids that, upon association with theprosthetic implant, form a solid or a gel in situ. Such controlledrelease vehicles are preferably associated with the prosthetic implantby one of the above-described methods. Preferred controlled releaseformulations are biodegradable (i.e., bioerodible).

One embodiment of the present invention relates to a method forproducing a prosthetic graft. Such a method comprises applying adherentcells to a porous prosthetic implant for containing blood in vivo,wherein the prosthetic implant has an outer surface and an inner surfacethat defines an interior space for containment of blood flow. Theadherent cells are applied to the outer surface of the prostheticimplant. As described above, the adherent cells are transformed with atleast one recombinant nucleic acid molecule operatively linked to atranscription control sequence, the recombinant nucleic acid moleculeencoding a protein that enhances patency of the prosthetic implant.

Preferably, the adherent cells are seeded (i.e., applied) onto the outersurface of the prosthetic implant so that they are uniformly dispersedon the surface of the implant (e.g., both radially and longitudinally,if the implant is a vessel). Such a uniform seeding has beendemonstrated by the present inventors to be sufficient to obtain thedesired protein production in vivo under blood shear stress conditions.In one embodiment, the number of cells seeded onto the outer surface ofa prosthetic implant is from about 1500 cells to about 4000 cells permm² surface area of the implant, and preferably, from about 2000 toabout 3500 cells per mm² surface area of the implant, and even morepreferably from about 2500 to about 3000 cells per mm² surface area ofthe implant. In another embodiment, the cells are seeded so that fromabout 0.5 μg to about 500 μg of each recombinant protein is secreted perml per mm² surface area of the implant. In one embodiment, the cells areseeded so that from about 0.5 μg to about 20 μg of each recombinantprotein is secreted per ml per mm² surface area of the implant, andpreferably, from about 1 μg to about 15 μg of each recombinant proteinis secreted per ml per mm² surface area of the implant. In anotherembodiment, the cells are seeded so that from about 1 μg to about 500 μgof each recombinant protein is secreted per ml per mm² surface area ofthe implant; and preferably, from about 10 μg to about 300 μg of eachrecombinant protein is secreted per ml per mm² surface area of theimplant.

The prosthetic implant can be seeded with the adherent cells by anymethod which allows the cells to naturally adhere to the implant (i.e.,without the need for exogenously added binding agents, sheaths or otherdevices), and which maintains cell viability and the ability of thecells to effectively express the protein. In one embodiment, the implantis seeded by applying cells to the implant on one side, rotating theimplant about 90°, seeding the next quadrant of the implant, andrepeating the procedure until all sections of the implant have beenseeded. The seeding can be performed manually, or using a programmablemechanical graft rotator. Since the cells are naturally adherent andwill not be exposed to blood shear forces, it is not necessary to usecomplex forced seeding methods that have been previously described forendothelial cell seeding, although such methods can be used, if desired.

Following application of the adherent cells to the implant to producethe prosthetic graft of the present invention, the graft is typicallyincubated under effective cell culture conditions as described above fora short time to allow for adequate cell adhesion to the implant. Thegraft can then be maintained under effective cell culture conditions asdescribed above until the graft is to be implanted into the recipient.The incubation period can be as short as about 5 minutes, or can beextended to at least about 14 days prior to implantation of the graftinto the host. Yet another advantage of the graft of the presentinvention is that once the graft is prepared, it can be maintained inculture until such time as the recipient is ready for implantation.

Yet another embodiment of the invention relates to a prosthetic graftfor containment of blood flow in vivo which includes: (a) a porousprosthetic implant for containing blood in vivo, having an outer surfacethat is not in contact with blood flow in vivo and an inner surface thatis in contact with blood flow in vivo, whereby the inner surface definesan interior space for containment of blood flow; and, (b) adherent cellsadhered to the outer surface of the porous prosthetic implant. In thisembodiment, the adherent cells can include both: (a) adherent cellswhich are transfected with a recombinant nucleic acid molecule thatencodes at least one protein that enhances patency of the prostheticimplant as described previously herein; and (b) adherent cells whichnaturally (i.e., endogenously) produce and secrete at least one proteinthat enhances patency of the prosthetic implant. As described above, anadherent cell includes cells that have been genetically modified (e.g.,by mutation or recombinant technology) to be adherent. Therefore, in oneaspect of this embodiment of the present invention, the adherent cellsare endothelial cells that have been genetically modified to beadherent. Endothelial cells naturally express proteins that enhancepatency of a prosthetic implant. As discussed above, an adherent cell iscapable of adhering to the outer surface of prosthetic implant withoutthe assistance of additional exogenous “binding factors”. Other aspectsof this embodiment of the present invention have been previouslydescribed herein as for the other embodiments of the present invention.

Another embodiment of the present invention is a method to implant aprosthetic graft for containment of blood flow into a patient in need ofsuch a graft. The method includes the step of implanting into arecipient patient a prosthetic graft of the present invention, suchgraft being configured and prepared as described in detail above. In oneembodiment of such a method, the method includes an initial step ofharvesting adherent cells, preferably fibroblast cells, from the patientwho is in need of a prosthetic graft for containing blood flow.

Preferably, the adherent cells used in the graft are autologous to thepatient. If the adherent cells are autologous, the cells are harvestedfrom the patient as discussed above, transfected with the desiredrecombinant nucleic acid molecules as described above, seeded onto theprosthetic implant as described above, and implanted into the patient.In one embodiment, the cells are selected from undifferentiated stemcell lines or embryonal cell lines. The cells can be induced to expressthe protein(s) which enhance patency either ex vivo, prior toimplantation of the graft into the patient, or in vivo, afterimplantation of the graft into the patient.

The step of implantation of the prosthetic graft of the presentinvention into a patient is/performed by any method which is suitablefor implantation of such a graft. The surgical techniques forimplantation of vascular grafts, artificial hearts, left ventricleassist devices, and dialysis shunts are well known and published in theart. Such methods are described, for example, in Persson and Griffey,1980, Surgical Clinics of North America 60(3):527-535; Mannick, 1979,Surgical Clinics of North America 59(4):581-596, incorporated herein byreference in their entireties.

If the prosthetic graft of the present invention includes adherent cellsthat are transformed with recombinant nucleic acid molecules withinducible promoters, the patient is administered, either before, during,or after the implantation of the graft, a compound which induces thepromoter to begin expression of the promoter at the outer surface of theprosthetic implant. Such compounds and promoters have been discussedpreviously herein. The compound can be administered in apharmaceutically acceptable carrier which is capable of maintaining thecompound in a form that, upon arrival of the compound at the graft site,the compound is biologically active such that induction of the promotercan occur. Examples of pharmaceutically acceptable carriers include, butare not limited to water, phosphate buffered saline, Ringer's solution,dextrose solution, serum-containing solutions, Hank's solution, otheraqueous physiologically balanced solutions, oils, esters, glycols,polymeric controlled release vehicles, biodegradable implants,liposomes, bacteria, viruses and cells. Routes of administration caninclude, but are not limited to, oral, nasal, topical, transdermal,rectal, and parenteral routes. Parenteral routes can include, but arenot limited to, subcutaneous, intradermal, intravenous, andintramuscular routes. The promoter-inducing compound can also be appliedto the graft or at a site adjacent to the graft at the time ofimplantation.

The following examples are provided for the purposes of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1

The following example describes the production of a prosthetic graftaccording to the present invention.

Primary Cell Culture:

Primary Rabbit Aortic Fibroblasts (RAF) were obtained from young maleWhite New Zealand rabbits. The aorta was explanted, the vessel was thenlongitudinally opened and the endothelium was removed by gently rubbingthe lumenal surface with a scraper. After this step, the vessel was cutinto small pieces, it was placed in a 60 mm dish containing 2 ml ofTrypsin and kept at 37° C. in a 5% CO₂ incubator for 60 minutes. Afterthis incubation, the vessel was removed and placed in 60 mm dishes with0.5 ml of DMEM supplemented with 10% FBS containing 2 mM L-glutamine and100 UI/mL Pen/Strep. After overnight incubation, 1.5 ml of culturemedium was added. RAFs were cultured with DMEM with 10% FBS, 2 mML-glutamine and 100 UI/mL Pen/Strep in a humidified 5% CO₂ atmosphere at37° C. Proliferating cells were used between passage 3 and 8 and wereused for infection when they reached 70% confluence. Rabbit fibroblastswere used for the preliminary in vitro assays described in Examples 2-4.

In the in vivo assay described in Example 5, pig primary fibroblastswere obtained from pig rectus fascia according to the followingprocedure. While the animal is under general anesthesia, the abdomen isaseptically prepared and under sterile conditions, a 10 cm longlongitudinal skin incision is made over the right rectus muscle. The fatunder the skin is gently divided and the anterior fascia of the rectusmuscle is exposed and cleaned of all fat tissue with a swab. The fasciais incised with a scalpel and a 5 cm×5 cm square of fascia is removedwith scissors. Hemostasis is performed, the surgical incision is closedin 2 layers, and anesthesia is terminated. Adipocytes were removed fromthe excised tissue by a forceps under surgical microscopy and the fasciawas cut in small pieces and digested with trypsin for 1 hour. Afterincubation, the pieces were then removed and placed in 60 mm dishes with0.5 ml of DMEM supplemented with 10% FBS containing 2 mM L-glutamine and100 UI/mL Pen/Strep. After overnight incubation, 1.5 ml of culturemedium was added.

Cell Infection with Adenovirus:

A similar protocol was used to infect both rabbit and pig fibroblasts.The virus used for this experiment was an adenovirus coding for VEGF.Cells were cultured in complete DMEM until 70% confluence. Prior theinfection, cells in one dish were harvested with trypsin and counted.Cells in the remaining dishes were infected with 200 pfu/cell andincubated in a humidified 5% CO₂ atmosphere at 37° C. for 90 minutes.The infected medium was replaced with D-MEM supplemented with 10% FBScomplete, and cells were kept in incubator at 37° C. for 24 hours. Toverify the infection efficiency, conditioned medium of infected cellsand conditioned medium of uninfected cells was collected and VEGFanalyzed in the medium was quantified with an ELISA assay commerciallyavailable from R&D performed according to standard procedures.

PhotoFix Preparation:

PhotoFix grafts stored in 50% ethanol were washed in PBS twice forfifteen minutes and twice for two hours in fresh PBS, then they wereplaced overnight at 4° C. in fresh PBS.

Seeding of PhotoFix:

After ethanol washing, the PhotoFix graft was cut in 5 cm pieces andseeded with infected cells at concentration of 1.25×10 cell/ml and twoml of suspension were used for seeding. After seeding on first quadrant,the PhotoFix was placed in a humidified 5% CO₂ atmosphere at 37° C. for30 minutes, then the PhotoFix was seeded on second quadrant and placedin a humidified 5% CO₂ atmosphere at 37° C. for 30 minutes. The twoother quadrants were seeded in the same way. At the end of thisprocedure the PhotoFix was incubated in a humidified 5% CO₂ atmosphereat 37° C. for 20 hours.

Example 2

The following example demonstrates that a prosthetic graft of thepresent invention produces the recombinant protein both inside andoutside of the graft under static in vitro culture conditions.

Production and secretion of VEGF under static in vitro conditions wasmeasured from the PhotoFix graft seeded perivascularly with rabbitaortic fibroblasts infected with AdV cmv VEGF as described in Example 1.Briefly, the seeded PhotoFix was mounted within a closed circuit placedin a chamber and connected to a peristaltic pump. Five ml of medium instatic condition were placed inside the PhotoFix, while 300 ml of mediumwas placed in the incubation chamber outside the PhotoFix. The chamberwas incubated under static conditions under shear stress in a humidified5% CO₂ atmosphere at 37° C. At the end of incubation, external andinternal medium was recovered (the internal medium was collected througha plastic outlet mounted on the circuit) and stored at −20° C. for theELISA assay, while the graft was fixed, dehydrated, embedded in paraffinand sectioned for histologic analyses.

After two days of −20° C. incubation, VEGF production was measured byenzyme-linked immunosorbant assay (ELISA) at given times (0, 12, 24, 48,72 hours) in the medium inside (internal) and outside (external) thegraft. The ELISA used was a commercially available ELISA kit for VEGFfrom RND. For each time point, a different graft was used for themeasurement. Negative and positive controls represent medium conditionedfor 24 hours by uninfected fibroblasts or infected fibroblasts,respectively.

The results of three separate experiments are illustrated in FIGS.1A-1C. FIG. 1D is a composite graph representing the average results ofall three experiments. FIGS. 1A-1D demonstrate that a prosthetic vesselgraft of the present invention produces significant and comparableamounts of VEGF protein both outside the graft and inside the graftunder in vitro static conditions.

Example 3

The following example demonstrates that a prosthetic graft of thepresent invention produces the recombinant protein both inside andoutside of the graft under shear stress in vitro culture conditions.

Release of VEGF under dynamic condition-shear stress of 1.5 dyn/cm²during in vitro conditions was measured from the PhotoFix graft seededperivascularly with rabbit aortic fibroblasts infected with AdV cmv VEGFas described in Example 1. Briefly, as described in Example 2 above, theseeded graft was mounted within a closed circuit placed in a chamber andconnected to a peristaltic pump. For the dynamic condition assays, 15 mlof medium were placed inside the PhotoFix and 300 ml of medium wasplaced in the incubation chamber outside the PhotoFix. The chamber wasincubated in a humidified 5% CO₂ atmosphere at 37° C. under dynamicconditions of shear stress by circulating the medium through the circuitat a velocity of 1.5 dyn/cm² as indicated. The medium was collected andtested for VEGF as described in Example 2.

VEGF production was measured by ELISA as described in Example 2 at giventimes (0, 4, 8, 12 hours) in the medium inside and outside the graft.For each time point, a different graft was used for the measurement.Negative and positive controls represent medium conditioned for 24 hoursby uninfected fibroblasts or infected fibroblasts, respectively.

The results of three separate experiments are illustrated in FIGS.2A-2C. FIG. 2D is a composite graph representing the average results ofall three experiments. FIGS. 2A-2D demonstrate that a prosthetic vesselgraft of the present invention produces significant and comparableamounts of VEGF protein both outside the graft and inside the graftunder in vitro dynamic shear stress of 1.5 dyn/cm², with the amountsincreasing over time.

Example 4

The following example demonstrates that a prosthetic graft of thepresent invention produces the recombinant protein both inside andoutside of the graft under shear stress in vitro culture conditions.

Release of VEGF under dynamic condition-shear stress of 10 dyn/cm²during in vitro conditions was measured from the PhotoFix graft seededperivascularly with rabbit aortic fibroblasts infected with AdV cmv VEGFas described in Example 1. Briefly, as described in Example 2 above, theseeded graft was mounted within a closed circuit placed in a chamber andconnected to a peristaltic pump. As described in Example 3, 15 ml ofmedium were placed inside the PhotoFix and 300 ml of medium was placedin the incubation chamber outside the PhotoFix. The chamber wasincubated in a humidified 5% CO₂ atmosphere at 37° C. under dynamicconditions of shear stress by circulating the medium through the circuitat a velocity of 10 dyn/cm² as indicated. The medium was collected andtested for VEGF as described in Example 2.

VEGF production was measured at given times (0, 4, 8, 12 hours) in themedium inside and outside the graft. For each time point, a differentgraft was used for the measurement. Negative and positive controlsrepresent medium conditioned for 24 hours by uninfected fibroblasts orinfected fibroblasts, respectively.

The results of three separate experiments are illustrated in FIGS.3A-3C. FIG. 3D is a composite graph representing the average results ofall three experiments. FIGS. 3A-3D demonstrate that, even under in vitrodynamic shear stress conditions of 10 dyn/cm², a prosthetic vessel graftof the present invention produces significant amounts of VEGF proteinboth outside the graft and inside the graft, with the amounts increasingover time.

Example 5

The following example demonstrates that a prosthetic graft of thepresent invention produces the recombinant protein in vivo.

In this study, fibroblasts were harvested from pig rectus muscle fasciaand cultured for 3 weeks as described in Example 1. The fibroblasts werethen transfected with the AdV cmv VEGF recombinant molecule, also asdescribed in Example 1, and the recombinant fibroblasts were seeded ontoPhotoFix grafts, also as described in Example 1. The grafts wereincubated overnight in DMEM medium in 5% CO₂ at 37° C., and the seededgraft was implanted in carotid position in the same pig from which thefibroblast cells were isolated. Briefly, after sedation with retanine, a30 kg domestic pig is anesthetized with halothane and mechanicallyventilated. The neck is aseptically prepared and under sterileconditions, a longitudinal cervical incision is made to expose thecommon carotid artery of one side. The common carotid artery is isolatedwith vessel loops, andheparin (100 U/kg) is given into the ear vein. Thecommon carotid artery is then clamped proximally and distally leavingabout 7 cm between the two clamps. A longitudinal arteriotomy is donedistally to the proximal clamp, and the proximal end of a 5 cm longPhotoFix graft is anastomosed to the arteriotomy with a continuoussuture of Prolene 6/0. A second arteriotomy is done proximally to thedistal clamp (towards the brain) and the other extremity of the PhotoFixgraft is sutured to the distal carotid as described before. Bothanastomoses are therefore done using the end to side technique. Theclamps are removed and hemostasis controlled with additional sutures, ifrequired. The segment of common carotid artery that has been bypassed isthen ligated proximally and distally and then removed. All blood thenflows through the graft. Blood specimens are taken from the carotidbefore the bypass (control) and then from the internal jugular vein onthe same side of the bypass, as blood passing into the graft and goingto the brain returns to the heart by way of the same side internaljugular vein. Blood specimens are taken at fixed time points. To followthe production of the protein of interest produced by the seededPhotoFix graft over long periods of time, a small polyethylene catheteris left inside the internal jugular vein and brought out to the skin byway of a subcutaneous tunnel. At the end of the procedure, the surgicalplanes are reapproximated and the animal is awakened and extubatedbefore returning the animal to its cage.

In this experiment, blood samples were taken from the ipsilateralinternal jugular vein before implant and then at 15 minutes, 30 minutes,45 minutes, 1 hour, 12 hours, 24 hours and 96 hours after implant. VEGFlevels were measured by ELISA as described in Example 2. FIG. 4demonstrates that detectable levels of VEGF were seen as soon as 45minutes after bypass (n.d.=non-detectable), and that such levelspersisted until the end of the study/(96 hours).

Example 6

The following example demonstrates that a prosthetic graft of thepresent invention produces the recombinant protein in vivo.

The experiment performed in Example 5 is repeated as described, but withthe following modifications.

1. The production of the protein VEGF is controlled and measured over alonger period of time, (1 week, 2 weeks, 4 weeks and 8 weeks).

2, A control graft, not seeded with fibroblasts, is implanted on theopposite side of the seeded graft in the same animal in one additionalanimal group, and in another additional animal group, a control graft(not seeded) is implanted in the absence of a seeded graft on theopposite side.

3. The graft is seeded with cell types selected from the group ofmesenchymal stem cells, bone marrow stem cells, embryonal stem cells,adipocytes, keratinocytes, vascular smooth muscle cells, platelets, orcells which have been genetically engineered to be adherent (e.g.,genetically modified endothelial cells).

4. The graft is seeded with cells transfected with a protein selectedfrom the group of vascular endothelial growth factor (VEGF),platelet-induced growth factor (PIGF), transforming growth factor β1(TGFβ1), acidic fibroblast growth factor (aFGF), basic fibroblast growthfactor (bFGF), transforming growth factor α (TGFα), epidermal growthfactor, osteonectin, angiopoietin 1 (Ang1), Ang2, insulin-like growthfactor (IGF), platelet-derived growth factor AA (PDGF-AA), PDGF-AB,PDGF-BB, tissue plasminogen activator (TPA), streptokinase, hirudin V,αv-βIII, urokinase plasminogen activator (uPA), nitric oxide synthase(NO synthase), or prostacyclin.

5. The graft is seeded with cells transfected alternate vectors whichexpress the protein of interest.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims:

1.-103. (canceled)
 104. A method of implanting a prosthetic graft forcontainment of blood flow into a patient, comprising: implanting intothe patient a prosthetic graft comprising (a) a porous prostheticimplant for containing blood in vivo, said prosthetic implant having anouter surface that is not in contact with blood flow in vivo and aninner surface that is in contact with blood flow in vivo, said innersurface defining an interior space for containment of blood flow, and(b) adherent cells adhered to the outer surface of said porousprosthetic implant, wherein said adherent cells are not seeded on theinner surface of said porous prosthetic implant, wherein said adherentcells are transfected with at least one recombinant nucleic acidmolecule operatively linked to a transcription control sequence, saidrecombinant nucleic acid molecule encoding a protein that enhancespatency of the prosthetic implant.
 105. The method of claim 104, furthercomprising, before the implanting step, harvesting fibroblast cells fromsaid patient.
 106. The method of claim 105, further comprisingtransfecting said fibroblast cells with an isolated nucleic acidmolecule encoding a protein that enhances patency of the graft.
 107. Themethod of claim 106, further comprising applying the transfectedfibroblast cells to the outer surface of said prosthetic implant. 108.The method of claim 104, wherein said adherent cells areundifferentiated stem cells.
 109. The method of claim 104, wherein saidadherent cells are embryonal cells.
 110. The method of claim 104,wherein said protein is selected from the group consisting of a proteinthat inhibits angiogenesis, a protein that inhibits thrombosis, aprotein that causes thrombolysis, a protein that inhibits smooth musclemigration or proliferation, and a vasodilator protein.
 111. The methodof claim 104, wherein the transcription control sequence is an induciblepromoter, and further comprising administering to the patient a compoundwhich induces the promoter.
 112. The method of claim 111, whereinadministering is performed via a route selected from the groupconsisting of oral, nasal, topical, transdermal, rectal, subcutaneous,intradermal, intravenous, and intramuscular.
 113. A method of implantinga prosthetic graft for containment of blood flow into a patient,comprising: implanting into the patient a prosthetic graft comprising(a) a porous prosthetic implant for containing blood in vivo, saidprosthetic implant having an outer surface that is not in contact withblood flow in vivo and an inner surface that is in contact with bloodflow in vivo, said inner surface defining an interior space forcontainment of blood flow, and (b) naturally adherent cells adhered tothe outer surface of said porous prosthetic implant, wherein saidnaturally adherent cells are not seeded on the inner surface of saidporous prosthetic implant, wherein said naturally adherent cells aretransfected with at least one recombinant nucleic acid moleculeoperatively linked to a transcription control sequence, said recombinantnucleic acid molecule encoding a protein, wherein said protein isexpressed by said cells ex vivo, wherein at least a portion of saidprotein, upon secretion from said cells, perfuses through pores of saidprosthetic implant to said inner surface of said prosthetic implant, andwherein said protein is selected from the group consisting of a proteinthat inhibits angiogenesis, a protein that inhibits thrombosis, aprotein that causes thrombolysis, a protein that inhibits smooth musclemigration or proliferation, and a vasodilator protein.
 114. The methodof claim 113, further comprising, before the implanting step, harvestingfibroblast cells from said patient.
 115. The method of claim 114,further comprising transfecting said fibroblast cells with an isolatednucleic acid molecule encoding a protein that enhances patency of thegraft.
 116. The method of claim 115, further comprising applying thetransfected fibroblast cells to the outer surface of said prostheticimplant.
 117. The method of claim 113, wherein said adherent cells areundifferentiated stem cells.
 118. The method of claim 113, wherein saidadherent cells are embryonal cells.
 119. The method of claim 113,wherein the transcription control sequence is an inducible promoter, andfurther comprising administering to the patient a compound which inducesthe promoter.
 120. The method of claim 119, wherein administering isperformed via a route selected from the group consisting of oral, nasal,topical, transdermal, rectal, subcutaneous, intradermal, intravenous,and intramuscular.